Primer for detection of human papillomavirus

ABSTRACT

Disclosed are primers specific to the genome of HPV genotypes 11, 16, 18 and 31. Also disclosed are a kit for detecting the HPV genome comprising the primers and a method of detecting the HPV genome using the primers.

TECHNICAL FIELD

The present invention relates to primers specific to the genome of humanpapillomavirus (hereinafter, referred to as “HPV”), a kit for detectingthe HPV genome comprising the primers, and a method of detecting the HPVgenome using the primers.

BACKGROUND ART

HPV is a double-stranded DNA virus whose circular genome isapproximately 8 kb long. HPV inhabits the vagina, and the infectionthereof is hard to treat and is not easily made to disappear. HPVinfects the epithelial cells of other mammals as well as humans, andgenerally induces warts, and sometimes malignant tumors, at the site ofinfection. HPV is detected in over 90% of condyloma accuminata cases(enlarged warts having a cauliflower-like appearance around the genitalsor the anus) and almost 100% of cervical cancer cases. In particular,cervical cancer accounts for 22.1% of all cancers found in women inKorea, and is the second leading cause of cancer death among women.

Thus, establishing a method of effectively detecting HPV, which causescervical cancer, is important for the diagnosis, prophylaxis and therapyof the disease. Also, HPV needs to be effectively detected to evaluatethe efficacy and toxicity of a vaccine against HPV after vaccination.

A nucleic acid-based test for diagnosing an infectious disease employs astandard method of isolating nucleic acids from individuals and clinicalmaterials. Since target DNA or RNA is present in clinical specimens insmall amounts, several major techniques used in diagnostic laboratoriesare based on signal amplification and target amplification. Thesemethods aid detection, are useful in the identification of individualswithout culture, and contribute to the treatment as well as diagnosis ofinfectious diseases. PCR, which is a nucleic acid amplificationtechnique (NAT), is widely used because it enables the selectiveamplification of specific targets, present in low concentrations, todetectable levels. In addition to the qualitative detection of viruses,quantitative determination of viral load in, clinical specimens is nowrealized to be of great importance with respect to the diagnosis,prognosis, and therapeutic monitoring of HPV infection (Pfaller M. A,Emer. Infect. Dis. 7, 2, 2001).

The genome of all types of HPV is divided into two major regions: earlyand late regions. The early region of about 4.5 kb codes for genes whichare associated with functions including viral DNA replication (E1),induction or suppression of the action of DNA encoding a proteininducing malignant transformation of host cells (E2), synthesis ofproteins responsible for the growth of host cells and viruses (E4),stimulation of the activity of epidermal growth factor (EGF) and colonystimulator factor (CSF) receptors (E5), and malignant transformationthrough permanent survival of cells, activation of oncogenes andinactivation of tumor suppressor genes (E7). In particular, theoncogenic E6 and E7 proteins, which are expressed after HPV infects theepithelial cells of a host, bind to tumor suppressor proteins of hostcells, p53 and pRB, respectively, thereby inhibiting the function of thetumor suppressor proteins, leading to the transformation of infectedcells, resulting in the development of tumors. The late region of 2.5 kbcomprises genes coding for viral major (L1) and minor (L2) capsidproteins and a non-coding region of 1 kb, which is called the longcontrol region (LCR) that regulates the transcription and translation ofthe two late genes.

With recent rapid advances in molecular biological techniques, thegenetic structure of HPV has been identified, revealing genomicsequences of many genotypes of HPV. HPV is classified according to thedifference in DNA sequences of E6, E7 and L1 open reading frames (ORFs).When the nucleotide sequences of the ORFs differ by more than 10%, anHPV is assigned a new genotype. HPV subtypes differ by 2% to 10%, andHPV variants differ by less than 2%.

In order to specifically detect high risk HPV types 16, 18 and 31 and alow risk HPV type 11 among a large number of HPV genotypes, which aredetected in tissues of cervical cancer and carcinoma in situ,respectively, the present inventors intended to detect a gene specificto each genotype of these viruses, and selected the L1 gene as such agene.

In order to specifically detect the HPV L1 gene, the present inventorsdetermined the sequences of L1 genes of the HPV types 11, 16, 18 and 31,which are specifically found in Koreans, and constructed primers capableof specifically binding to the L1 gene of each HPV type. The presentinventors found that when PCR was performed with the primers, each HPVgenotype can be specifically detected and can be precisely quantifieddown to very low amounts, thereby leading to the present invention.

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide a primerpair selected from among pairs of primers capable of complementarilybinding to the genome of human papillomavirus (HPV) and havingnucleotide sequences represented by SEQ ID Nos. 1 and 2, SEQ ID Nos. 3and 4, SEQ ID Nos. 5 and 6, and SEQ ID Nos. 7 and 8.

It is another object of the present invention to provide a method ofdetecting the HPV genome, which is based on performing a polymerasechain reaction (PCR) for DNA contained in a biological sample using oneor more primer pairs selected from among pairs of primers capable ofcomplementarily binding to the HPV genome and having nucleotidesequences represented by SEQ ID Nos. 1 and 2, SEQ ID Nos. 3 and 4, SEQID Nos. 5 and 6, and SEQ ID Nos. 7 and 8.

It is a further object of the present invention to provide a kit fordetecting the HPV genome, comprising one or more primer pairs selectedfrom among pairs of primers capable of complementarily binding to theHPV genome and having nucleotide sequences represented by SEQ ID Nos. 1and 2, SEQ ID Nos. 3 and 4, SEQ ID Nos. 5 and 6, and SEQ ID Nos. 7 and8.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 schematically represents the construction of recombinant plasmidsconstructed with amplified L1 genes of HPV genotypes 11, 16, 18 and 31(A), and also shows the results of restriction enzyme mapping of therecombinant plasmids (B);

FIG. 2 is an alignment of HPV 16 L1 sequences;

FIG. 3 is an alignment of HPV 31 L1 sequences;

FIG. 4 is an alignment of HPV 11 L1 sequences;

FIG. 5 is an alignment of HPV 18 L1 sequences;

FIG. 6 is a multiple alignment of nucleotide sequences of L1 genes ofHPV genotypes 11, 16, 18 and 31 and the consensus L1 sequence;

FIG. 7 shows the results of a sensitivity test using plasmid DNAtemplates, each of which carry the HPV 11, 16, 18 or 31 L1 gene. FIG. 7Ashows measured band intensity of separated PCR products on agarose gel.FIG. 7B shows band intensity measured using the software Quantity One(Bio-Rad), and the relationship between band intensity and the number ofplasmid copies;

FIG. 8 shows the results of a differentiality test using plasmid DNAtemplates, each of which carries the HPV 11, 16, 18 or 31 L1 gene;

FIG. 9 shows the results of heat stability and long-term preservationtests for HPV L1 plasmids after storage for 3 weeks;

FIGS. 10 and 11 show the results of heat stability and long-termpreservation tests for HPV L1 plasmids after storage for 15 weeks; and

FIG. 12 shows the results of an applicability test using predeterminedamounts of HPV L1 plasmids supplemented with various DNA backgrounds.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to primers capable ofcomplementarily binding to the HPV genome.

In a detailed aspect, the present invention relates to a primer pairselected from among pairs of primers capable of complementarily bindingto the HPV genome and having the nucleotide sequences represented by SEQID Nos. 1 and 2, SEQ ID Nos. 3 and 4, SEQ ID Nos. 5 and 6, and SEQ IDNos. 7 and 8.

The term “primer”, as used herein, refers to a short nucleic acidsequence having a free 3′ hydroxyl group, which is able to undergobase-pairing interaction with a complementary template and serves as astarting point for replicating the template strand. A primer is able toinitiate DNA synthesis in the presence of a reagent for polymerizationand four different nucleoside triphosphates in suitable buffers and at asuitable temperature. With respect to the objects of the presentinvention, primers specifically amplify a specific region of the L1 geneof HPV 16, described in FIG. 2, HPV 31, described in FIG. 3, HPV 11,described in FIG. 4, and HPV 18, described in FIG. 5. Thus, the primersof the present invention consist of a pair of sense and antisenseprimers having a sequence of 7 to 50 nucleotides, and more preferably 10to 30 nucleotides, the sequence capable of complementarily binding tothe aforementioned HPV L1 gene. In detail, a specific region of the HPVL1 gene may be specifically amplified with a pair of primers having thenucleotide sequences of SEQ ID Nos. 1 and 2 for HPV 11, a pair ofprimers having the nucleotide sequences of SEQ ID Nos. 3 and 4 for HPV16, a pair of primers having the nucleotide sequences of SEQ ID Nos. 5and 6 for HPV 18, and a pair of primers having the nucleotide sequencesof SEQ ID Nos. 7 and 8 for HPV 31.

The primers of the present invention may be chemically synthesized usinga phosphoramidite solid support method or other widely known methods.These nucleic acid sequences may also be modified using any means knownin the art. Non-limiting examples of such modifications includemethylation, capsulation, replacement of one or more native nucleotideswith analogues thereof, and inter-nucleotide modifications, for example,modifications to uncharged conjugates (e.g., methyl phosphonate,phosphotriester, phosphoroamidate, carbamate, etc.) or chargedconjugates (e.g., phosphorothioate, phosphorodithioate, etc.). Nucleicacids may contain one or more additionally covalent-bonded residues,which are exemplified by proteins (e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), intercalating agents (e.g.,acridine, psoralene, etc.), chelating agents (e.g., metals, radioactivemetals, iron, oxidative metals, etc.), and alkylating agents. Thenucleic acid sequences of the present invention may also be alteredusing a label capable of directly or indirectly supplying a detectablesignal. Examples of such a label include radioisotopes, fluorescentmolecules, and biotin.

When PCR was performed with the primers provided in the presentinvention, which have the nucleotide sequences of SEQ ID Nos. 1 and 2,SEQ ID Nos. 3 and 4, SEQ ID Nos. 5 and 6, and SEQ ID Nos. 7 and 8, theprimers were found to be able to specifically detect each of the fourdifferent HPV genotypes and to be sensitive enough to amplify as few as62.5 copies of a plasmid.

Thus, the present primers may be useful in the detection of HPVinfections, the identification of infective HPV genotypes, theepidemiological evaluation of HPV, the effectiveness and toxicity ofdeveloped HPV vaccines, and the like.

In another aspect, the present invention provides a method of detectingthe HPV genome, which is based on performing a polymerase chain reaction(PCR) for DNA contained in a biological sample using one or more primerpairs selected from among pairs of primers capable of complementarilybinding to the HPV genome and having the nucleotide sequencesrepresented by SEQ ID Nos. 1 and 2, SEQ ID Nos. 3 and 4, SEQ ID Nos. 5and 6, and SEQ ID Nos. 7 and 8.

The term “biological sample”, as used herein, includes, but is notlimited to, samples, such as tissues, cells, whole blood, sera, plasma,saliva, sputa, cerebrospinal fluid, urine, or the like, of individualsinfected with HPV or suspected of being infected with HPV, orindividuals vaccinated with a HPV vaccine.

A method for identifying the presence and genotype of HPV isparticularly not limited as long as it employs the aforementionedprimers. Examples of such methods include direct identification of HPVDNA using a primer of a specific strand as a probe, Southern blotting,dot blotting, and filter in situ hybridization (FISH). Alternativemethods include a method based on amplifying HPV DNA using a pair ofprimers, genotype-specific polymerase chain reaction (PCR), andgeneral-primer PCR. PCR is most preferred.

The term “polymerase chain reaction (PCR)”, as used herein, is arepresentative nucleic acid amplification technique (NAT), whichenzymatically amplifies a specific DNA region of interest in vitro. ThePCR method, which was developed in 1985 by Mullis et al., can amplifyany segment of a DNA molecule if its boundary sequences are known. PCRbasically consists of three major steps: denaturation, annealing andextension. A specific DNA sequence is amplified while these three stepsare repeated. In the first step (denaturation) of PCR, a double-strandedtemplate DNA is denatured into two single strands. In the second step(annealing), primers anneal with the two kinds of single-stranded DNA,in which a sequence desired to be amplified is interposed between theprimer binding regions. In the third step (extension), a heat-resistantDNA polymerase extends the primers and synthesizes the complementarystrand of the target sequence. This cycle is repeated 25 to 30 times.

Primers are the most important factor determining the reliability of PCRresults. Some primer sequences can give rise to non-specificamplification, leading to false results. In this regard, the presentinvention provides reliable primer pairs. The performance of PCR withthe primer pairs of the present invention enables accurate detection ofHPV genotypes and sensitive quantitative analysis of very small amounts.Also, when PCR is carried out with the primer pairs of the presentinvention, consistent results are obtained upon repeated PCRperformance. That is, since the primer pairs of the present inventionare highly valid and reliable, the results obtained with the presentprimer pairs are highly reliable.

In a preferred aspect, the present invention provides a method ofdetecting the HPV 11 L1 gene using a pair of primers having thenucleotide sequences of SEQ ID Nos. 1 and 2.

In another preferred aspect, the present invention provides a method ofdetecting the HPV 16 L1 gene using a pair of primers having thenucleotide sequences of SEQ ID Nos. 3 and 4.

In a further preferred aspect, the present invention provides a methodof detecting the HPV 18 L1 gene using a pair of primers having thenucleotide sequences of SEQ ID Nos. 5 and 6.

In yet another preferred aspect, the present invention provides a methodof detecting the HPV 31 L1 gene using a pair of primers having thenucleotide sequences of SEQ ID Nos. 7 and 8.

A PCR for amplifying an HPV gene, in detail the L1 gene, using theprimers of the present invention may be carried out through an ordinaryPCR method. Also, conditions including time, temperature and cyclenumber, under which denaturation, annealing and extension reactions areallowed to occur, may vary. In the present invention, PCR conditionsincluded 35 cycles of denaturation at 94° C. for 1 min, annealing at 51°C. for 1 min, and extension at 72° C. for 1 min or 1 min 30 sec.

In a further aspect, the present invention provides a kit for detectingthe HPV genome, comprising one or more primer pairs selected from amongpairs of primers capable of complementarily binding to the HPV genomeand having the nucleotide sequences represented by SEQ ID Nos. 1 and 2,SEQ ID Nos. 3 and 4, SEQ ID Nos. 5 and 6, and SEQ ID Nos. 7 and 8.

In addition to the primer pairs, the detection kit of the presentinvention is composed of one or more different compositions, solutionsor instruments, which are suitable for analysis methods. Preferably, thekit of the present invention includes the following constituents: acontainer containing detection primers; amplification reaction tubes orother suitable containers; reaction buffer (pH and magnesiumconcentration of which may vary); dNTPs; an enzyme such asTaq-polymerase; RNase; and sterile water. More preferably, the kit mayfurther include a plasmid carrying an HPV gene as a positive control inorder to realize quantitative analysis. Such a plasmid may be one ormore selected from among pGEM-HPV11 L1, pGEM-HPV16 L1, pGEM-HPV18 L1,and pGEM-HPV31 L1, which will be described in the following examples.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

EXAMPLE 1 Construction of Recombinant HPV L1 Plasmids (Standard DNA)

PCR primers specific to low risk HPV 11 and high risk HPV 16, 18 and 31were designed based on major protein (HPV L1) sequences of the differentgenotypes of HPV, which are deposited in GenBank. In order to obtain HPVgenotypes commonly found in Korean, tissues of Korean cervical cancerpatients were obtained from clinical hospitals, and genomic DNA as anHPV genome source was extracted from the tissues. Biological tissuesamples were paraffin sections or biopsy samples prepared forpathological examination. PCR was carried out using the extractedgenomic DNA with primers having the nucleotide sequences of SEQ ID Nos.9 to 16, which are listed in Table 1, below. As a result, PCR productsof about 1.6 kb were obtained.

TABLE 1 Primer sequences against the HPV L1 gene for the preparation ofnational (Korean) standard DNA PCR HPV fragment genotype length PCRprimer sequence HPV 16 1596 bp Sense 5′-GCCCCCAAGCTTGCCGCCACCATGCAGGTGACTTTTATTTACATCC-3′ (SEQ ID No. 9) Anti-5′-ATCGGGCTCGAGCAGCTTACGTTT sense TTTGCGTTTAGC-3′ (SEQ ID No. 10) HPV 181707 bp Sense 5′-GCCCCCAAGCTTGCCGCCACCATG TGCCTGTATACACGG-3′ (SEQ ID No.11) Anti- 5′-ATCGGGGAATTCCTTCCTGGCACG sense TACACGCACACG-3′ (SEQ ID No.12) HPV 31 1515 bp Sense 5′-GCCCCCAAGCTTGCCGCCACCATGTCTCTGTGGCGGCCTAGC-3′ (SEQ ID No. 13) Anti- 5′-ATCGGGGAATTCCTTTTTAGTTTTsense TTTACGTTTTGCTGGTGTAGTGG-3′ (SEQ ID No. 14) HPV 11 1506 bp Sense5′-GCCCCCAAGCTTGCCGCCACCATG TGGCGGCCTAGCGACAGC-3′ (SEQ ID No. 15) Anti-5′-ATCGGGGAATTCCTTTTTGGTTTT sense GGTACGTTTTCGTTTGGG-3′ (SEQ ID No. 16)

PCR was carried out under the following conditions. PCR was carried outusing the DNA samples extracted from tissues from patients as templateswith 2.5 mM dNTP, reaction buffer, primer pairs (20 pmol) listed inTable 1, and SuperTaq Plus. According to the optimal annealingtemperature of primers, a cycle of denaturation at 94° C. for 1 min,annealing at Ta for 1 min, and extension at 72° C. for 1 min 30 sec wasrepeated thirty five times, followed by final extension at 72° C. for 10min. Each PCR product was cloned into pGEM-T-Easy vector (Promega, USA)and transformed into E. coli DH5α. Plasmid DNA was then isolated anddigested with EcoRI to determine whether the PCR product wassuccessfully inserted (FIG. 1).

The nucleotide sequences of the PCR products were determined andcompared with previously known nucleotide sequences coding for the L1protein of HPV genotypes. HPV 16 was compared with AF402678, HPV 31 withJ04353, HPV 11 with NC_(—)001525, and HPV 18 with NC_(—)001357. As aresult, the nucleotide sequences coding for the L1 protein of the fourHPV genotypes, which were identified according to the proceduredescribed above, were found to be highly similar to the conventionallyknown nucleotide sequences coding for L1 proteins of the HPV genotypes.

EXAMPLE 2 Large Preparation and Quantification of the Recombinant HPV L1Plasmids

E. coli cells transformed with the recombinant HPV L1 plasmids preparedin Example 1 were inoculated in 10 ml of LB medium supplemented withampicillin, and grown in a shaking incubator at 37° C. overnight.Plasmid DNA was then isolated using an alkaline lysis method, andprecisely quantified using a spectrophotometer. The plasmid copy numberwas calculated according to Equation 1, below.Copy number of 1 kb fragment=(1000 bp×660 g/mole)/(6.023×10²³molecules)=1×10⁻¹⁸ g (1 fg)  [Equation 1]

Equation 1 means the following. One copy of a 1 kb (1000 bp) plasmidweighs 1×10⁻¹⁸ g (1 fg), and one gram of 1 kb plasmid DNA contains 10¹⁸copies of the plasmid.

Using Equation 1, the number of copies of each of different HPV genotypeplasmids was calculated, and a plasmid solution having 2000 copies wasserially diluted two-fold, thereby yielding 10-μl solutions containing2000, 1000, 500, 250, 125 and 62.5 copies of the plasmids.

EXAMPLE 3 Evaluation of the Sensitivity of Specific Primers Using theRecombinant HPV L1 Plasmids

Using the 10 μl plasmid solutions containing 2000, 1000, 500, 250, 125and 62.5 copies, prepared in Example 2, PCR was carried out. As aresult, PCR primers displayed sensitivity in a manner dependent on thenumber of plasmid copies (indicating that the 10 μl DNA solutionsrespectively contained 2000, 1000, 500, 250, 125 and 62.5 copies of theplasmids). Referring to FIG. 6, PCR primers represented by SEQ ID Nos. 1to 8 (Table 2) were determined.

TABLE 2 Primers for detecting the HPV L1 gene Genotype Sense primerAnti-sense primer HPV 11 TTAGGCGTTGGTGTTAGTGG AAAATTCATAGCACCAAAGC (SEQID No. 1) (SEQ ID No. 2) HPV 16 TTAGGTGTGGGCATTAGTGGAAAGTCCATAGCACCAAAGC (SEQ ID No. 3) (SEQ ID No. 4) HPV 18TTAGGTGTTGGCCTTAGTGG AAAGTCCATGGCACCATATC (SEQ ID No. 5) (SEQ ID No. 6)HPV 31 TTAGGTGTAGGTATTAGTGG AAAATCCATAGCTCCAAAGC (SEQ ID No. 7) (SEQ IDNo. 8)

PCR was carried out as follows. 5 μl of 2.5 mM dNTP was mixed with 5 μlof 10× buffer, primers (20 pmol) of SEQ ID Nos. 1 to 8, 0.5 μl of Taqpolymerase, and distilled water to give a final volume of 40 μl. Themixture was supplemented with 10 μl of each template, thereby yielding aPCR mixture. PCR conditions included 35 cycles of denaturation at 94° C.for 1 min, annealing at 51° C. for 1 min, and extension at 72° C. for 1min, followed by final extension at 72° C. for 10 min. PCR products werethen separated on a 1.5% agarose gel for 40 min, and stained withethidium bromide (EtBr). Band intensity was measured using the software,Quantity One (Bio-Rad). Then, a regression function was derived in orderto determine the relationship between band intensity and the number ofplasmid copies, and a relative coefficient R was calculated to determinewhether it was greater than 0.9. As a result of the sensitivity test forthe PCR amplification method, the band intensity, as shown in FIG. 7,decreased in all of the four HPV genotypes in a manner dependent on thenumber of plasmid copies, and this PCR method was found to have asensitivity detecting as few as 62.5 copies of the HPV L1 plasmids. WhenDNA was run on an agarose gel to determine the relationship between bandintensity and the number of plasmid copies, the relative coefficient, asshown in FIG. 7, was greater than 0.9. These results indicate that aplasmid copy number test using the method of the present inventionprovides reliable results.

EXAMPLE 4 Evaluation of the Specificity of Primers Using the RecombinantHPV L1 Plasmids

In order to determine whether the primers used in Example 3 specificallyamplify each HPV genotype, the primer sets to the four different HPVgenotypes were evaluated for whether they differentially amplify L1templates of different HPV genotypes. PCR was carried out under the sameconditions as in Example 3 except that the templates were used in aconcentration of 1000 copies. PCR was performed with each primer setusing each of the four different HPV genotypes as a template. As aresult of the PCR with each primer set using 1000 copies of each HPVgenotype as a template, all primer sets to HPV 11, 16, 18 and 31 werefound to specifically amplify only their corresponding templates (FIG.8). The results, specifically that the primers of the present inventionprecisely detect only their corresponding HPV genotypes under optimizedPCR conditions, indicate that the different primer sets enable HPVgenotyping of clinical samples and differential detection of high riskHPV types 11, 16 and 18, infections of which are most likely to progressto cervical cancer. Thus, the present primers may become a very usefulmeans of clinical diagnosis.

EXAMPLE 5 Evaluation of the Heat Stability and Long-Term Preservation ofthe Recombinant HPV L1 Plasmids

To evaluate the heat stability and long-term preservation of primers,first, 30 μl of 1000 copies of each HPV L1 plasmid was aliquotted intofifteen DNase/RNase-free vials. The containers were stored at 4° C., 22°C. and 37° C. for a heat stability test, and at −80° C. for long-termstorage. Every three weeks, one vial at each storage temperature wassubjected to a sensitivity test, which was carried out according to thesame procedure as in Example 3. The PCR with plasmids stored at 22° C.and 37° C. for three weeks showed negative results, indicating thatstandard DNA has a very low stability when stored at 22° C. and 37° C.(FIG. 9). In contrast, standard DNA stored at 4° C. and −80° C. for 15weeks still provided highly sensitive PCR results, indicating that thestandard DNA of the present invention is stable when stored at −80° C.for a long period of time (FIGS. 10 and 11).

EXAMPLE 6 Evaluation of the Applicability of Primers Using theRecombinant HPV L1 Plasmids

The procedure with the HPV L1 templates and primers provided in thepresent invention was evaluated to determine whether it could detect theHPV genome in human clinical samples, as follows. Genomic DNA wasextracted from human rhabdomyosarcoma (RD), HeLa and SLK cells using agenomic DNA extraction kit (Qiagen), and the concentration thereof wasdetermined using a spectrophotometer. Each DNA solution was diluted toconcentrations of 10 ng and 100 ng. 100 μl of each dilution wasaliquotted and stored at −20° C. Then, PCR was carried out with 100 ngand 1 μg of genomic DNA background using the same templates as in thesensitivity test of Example 3. PCR products were analyzed according tothe same method used for the sensitivity test. In the PCR with thegenomic DNA background, the HPV templates, as shown in FIG. 12, could bedetected even with as few as 62.5 copies, as in the absence of humangenomic DNA. The PCR with genomic DNA background from HeLa cells, whichcontain the HPV 18 genome, exhibited positive results in all lanes.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the primer pairs specific to the L1 gene ofHPV 11, 16, 18 and 31 according to the present invention may be usefulin the detection of HPV infections, the identification of infected HPVgenotypes, evaluation of the effectiveness and toxicity of developed HPVvaccines, and the like.

1. A method of selectively detecting human papillomavirus (HPV)genotypes, comprising performing a polymerase chain reaction for genomicDNA contained in a biological sample using each of the primer pairshaving nucleotide sequences represented by SEQ ID Nos. 1 and 2, SEQ IDNos. 3 and 4, SEQ ID Nos. 5 and 6, and SEQ ID Nos. 7 and 8.